Aldose reductase inhibitors and uses thereof

ABSTRACT

The present invention relates to novel compounds and pharmaceutical compositions thereof, and methods for promoting healthy aging of skin, the treatment of skin disorders, the treatment of cardiovascular disorders, the treatment of renal disorders, the treatment of angiogenesis disorders, such as cancer, treatment of tissue damage, such as non-cardiac tissue damage, the treatment of evolving myocardial infarction, and the treatment of various other disorders, such as complications arising from diabetes with the compounds and compositions of the invention. Other disorders can include, but are not limited to, atherosclerosis, coronary artery disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, infections of the skin, peripheral vascular disease, stroke, and the like.

This application is a continuation of U.S. patent application Ser. No.15/990,121 filed May 25, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/489,832 filed Apr. 18, 2017, which is acontinuation of U.S. patent application Ser. No. 14/541,365, filed Nov.14, 2014 and issued as U.S. Pat. No. 9,650,383 on May 16, 2017, whichwas filed as a divisional of U.S. patent application Ser. No.13/742,573, filed Jan. 16, 2013 and issued as U.S. Pat. No. 8,916,563 onDec. 23, 2014, which was filed as a continuation-in-part ofInternational Application Number PCT/US2011/044038, filed Jul. 14, 2011,which claims priority to U.S. Provisional Application No. 61/365,098,filed Jul. 16, 2010, the contents of each of which are herebyincorporated by reference in their entireties.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

GOVERNMENT SUPPORT

This invention was made with government support under grant RR024156awarded by the NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel compounds and pharmaceuticalcompositions thereof, and methods for promoting healthy aging of skin,the treatment of skin disorders, the treatment of cardiovasculardisorders, the treatment of renal disorders, the treatment ofangiogenesis disorders, such as cancer, treatment of tissue damage, suchas non-cardiac tissue damage, the treatment of evolving myocardialinfarction, and the treatment of various other disorders, such ascomplications arising from diabetes with the compounds and compositionsof the invention. Other disorders can include, but are not limited to,atherosclerosis, coronary artery disease, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, infections of the skin, peripheralvascular disease, stroke, and the like.

BACKGROUND OF THE INVENTION

Diabetes is one of the most common chronic disorders, in which highblood glucose levels result from a lack of insulin production and/orinsulin sensitivity. Individuals with high blood glucose metabolize moreglucose via a glucose to sorbitol to fructose pathway in insulininsensitive cells such as lenses, peripheral nerves and glomerulus. Thisleads to an overabundance of sorbitol in the cells, which is not easilydiffused through the cell membrane. The increased concentration ofsorbitol triggers an influx of water into the cells, causing swellingand potential damage.

Aldose reductase, an enzyme present in many parts of the body, catalyzesthe reduction of glucose to sorbitol, one of the steps in the sorbitolpathway that is responsible for fructose formation from glucose. Aldosereductase activity increases as the glucose concentration rises indiabetic conditions where tissues are no longer insulin sensitive. Thesetissues include, for example, lenses, peripheral nerves and glomerulusof the kidney. Sorbitol cannot easily diffuse through cell membranes andtherefore accumulates, causing osmotic damage, which in turn leads toretinopathy, neuropathy, and nephropathy. Therefore, inhibition ofaldose reductase could prevent the buildup of sorbitol in insulininsensitive cells in diabetics, and presents a novel method to preventthe macrovascular and microvascular complications in diabetic patients.In addition, aldose reductase inhibitors, such as zopolrestat, may aidin treating or ameliorating such effects and have shown efficacy inwound healing in the corneal epithelium of diabetic animal models.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to compounds of formula(I)

wherein,

R¹ is H, (C₁-C₆)-alkyl, (C₁-C₆)-hydroxyalkyl, or (C₁-C₆)-aminoalkyl;

X¹ is N or CR³;

X² is N or CR⁴;

X³ is N or CR⁵;

X⁴ is N or CR⁶; with the proviso that two or three of X¹, X², X³, or X⁴are N;

Y is a bond, C═O, C═S, C═NH, or C═N(C₁-C₄)-alkyl;

Z is

A¹ is NR¹¹, O, S or CH₂;

A² is N or CH;

A³ is NR¹¹, O, or S;

R³ through R¹⁰ are independently hydrogen, halogen, cyano, acyl,haloalkyl, haloalkoxy, haloalkylthio, trifluoroacetyl, (C₁-C₄)-alkyl,(C₁-C₄)-alkoxy, (C₁-C₄)-alkylthio, (C₁-C₄)-alkylsulfinyl, or(C₁-C₄)-alkylsulfonyl; or two of R³ through R⁶ or two of R⁷ through R¹⁰taken together are (C₁-C₄)-alkylenedioxy; and

R¹¹ is hydrogen, C₁-C4 alkyl, or C(O)O-(C1-C₄)-alkyl; orpharmaceutically acceptable salts or solvates thereof

In another aspect, the present invention is also directed topharmaceutical compositions comprising a compound of formula (I), or apharmaceutically acceptable salt or solvent thereof

In yet another aspect, the present invention is also directed to methodsof treatment comprising administration of a compound of formula (I) or apharmaceutically acceptable salt or solvent thereof, or pharmaceuticalcompositions comprising a compound of formula (I), or a pharmaceuticallyacceptable salt or solvent thereof, to a subject in need thereof. Thecompounds and/or compositions of the invention may be useful, forexample, in promoting healthy aging of skin, the treatment of skindisorders, the treatment of angiogenesis disorders, such as cancer, thetreatment of tissue damage, the treatment of cardiovascular disorders,the treatment of renal disorders, the treatment of evolving myocardialinfarction, the treatment of various other disorders, such ascomplications arising from diabetes. Such disorders can include, but arenot limited to, atherosclerosis, coronary artery disease, diabeticnephropathy, diabetic neuropathy, diabetic retinopathy, infections ofthe skin, peripheral vascular disease, stroke, and the like.

In still another aspect, the invention is directed to processes forpreparing compounds of formula (I).

The present invention is based, in part, on certain discoveries whichare described more fully in the Examples section of the presentapplication. For example, the present invention is based, in part, onthe discovery of compounds of formula (I) and the aldose reductaseinhibition exhibited by such compounds.

These and other embodiments of the invention are further described inthe following sections of the application, including the DetailedDescription, Examples, and Claims. Still other objects and advantages ofthe invention will become apparent by those of skill in the art from thedisclosure herein, which are simply illustrative and not restrictive.Thus, other embodiments will be recognized by the ordinarily skilledartisan without departing from the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a solubility curve for Compound A and zopolrestat in aqueoussolution.

FIG. 2 shows aldose reductase inhibitory activity for Compound A andzopolrestat.

FIG. 3 shows the concentration versus inhibition of aldose reductasecurve for Compound A.

DETAILED DESCRIPTION OF THE INVENTION

Aldose reductase inhibitors are described, for example, in U.S. Pat.Nos. 5,677,342; 5,155,259; 4,939,140; U.S. patent application Ser. No.11/210,283; and Roy et al., in Diabetes Research and Clinical Practice,Vol. 10, Issue 1, 91-97; and references cited therein; each of whichhereby incorporated by reference in its entirety. Aldose reductaseinhibitors include, for example, zopolrestat, epalrestat, ranirestat,berberine and sorbinil. A novel family of aldose reductase inhibitorshas been discovered and is described herein. Surprisingly, this novelfamily comprises compounds that exhibit dramatically improved propertiessuch as, for example, binding affinity, solubility and polarity relativeto other aldose reductase inhibitors such as, for example, zopolrestat.Compounds such as zopolrestat are described, for example in U.S. Pat.Nos. 4,939,140; 6,159,976; and 6,570,013; each of which herebyincorporated by reference in its entirety. The inventors have alsosurprisingly discovered that changes in functionalities at positionsthat often reside in a hydrophobic binding pocket of the enzyme do notabolish binding of the compounds to the enzyme. For example,incorporation of a polar moiety such as, for example, a nitrogen atom inthe phenyl ring of the phthalazine, results in improvement of bindingaffinity and solubility. This is unexpected, in part due to thepropensity of the phenyl ring of the phthalazine to occupy a hydrophobicpocket in the enzyme.

The compounds and/or compositions of the invention may be effective intreating, reducing, and/or suppressing complications related to aldosereductase activity such as, for example, neuropathy, retinopathy,nephropathy and multiple complications in diabetic patients. Thecompounds and/or compositions of the invention may also be effective intreating, reducing, and/or reducing cardiovascular and renal disordersin non-diabetic patients, as well as promoting healthy aging of skin orwound healing.

Abbreviations and Definitions

The term “aldose reductase inhibitor” refers to compounds and salts orsolvates thereof that function by inhibiting the activity of the enzymealdose reductase, which is primarily responsible for regulatingmetabolic reduction of aldoses. Exemplary aldoses include, but are notlimited to, glucose or galactose, and their corresponding polyols, suchas sorbitols and galactitols. Exemplary aldose reductase inhibitors maybe found in U.S. Pat. Nos. 4,939,140; 4,954,629; and 5,304, 557; each ofwhich hereby incorporated by reference in its entirety.

The term “compound of the invention” as used herein means a compound offormula (I). The term is also intended to encompass salts, hydrates,pro-drugs and solvates thereof

The term “composition(s) of the invention” as used herein meanscompositions comprising a compound of the invention, and salts,hydrates, pro-drugs, or solvates thereof. The compositions of theinvention may further comprise other agents such as, for example,excipients, stabilants, lubricants, solvents, and the like.

The term “alkyl”, as used herein, unless otherwise indicated, refers toa monovalent aliphatic hydrocarbon radical having a straight chain,branched chain, monocyclic moiety, or polycyclic moiety or combinationsthereof, wherein the radical is optionally substituted at one or morecarbons of the straight chain, branched chain, monocyclic moiety, orpolycyclic moiety or combinations thereof with one or more substituentsat each carbon, where the one or more substituents are independentlyC₁-C₁₀ alkyl. Examples of “alkyl” groups include methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, tent-butyl, pentyl, hexyl,heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,norbornyl, and the like.

The term “halogen”, as used herein, means chlorine (Cl), fluorine (F),iodine (I) or bromine (Br).

The term “method(s) of the invention” as used herein means methodscomprising treatment with the compounds and/or compositions of theinvention.

The term “solvate” as used herein means a compound, or apharmaceutically acceptable salt thereof, wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent is physiologically tolerable at the dosage administered.Examples of suitable solvents are ethanol, water and the like. Whenwater is the solvent, the molecule is referred to as a “hydrate.”

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or pharmaceutically acceptable salts,solvates, pro-drugs or hydrates thereof, with other chemical components,such as physiologically acceptable carriers and excipients. The purposeof a pharmaceutical composition is to facilitate administration of acompound to an organism or subject.

A “pro-drug” or “pro-drug” refers to an agent which is converted intothe parent drug in vivo. Pro-drugs are often useful because, in somesituations, they are easier to administer than the parent drug. They arebioavailable, for instance, by oral administration whereas the parentdrug is either less bioavailable or not bioavailable. The pro-drug alsohas improved solubility in pharmaceutical compositions over the parentdrug. For example, the compound carries protective groups which aresplit off by hydrolysis in body fluids, e.g., in the bloodstream, thusreleasing active compound or is oxidized or reduced in body fluids torelease the compound. The term “pro-drug” may apply to suchfunctionalities as, for example, the acid functionalities of thecompounds of formula I. Pro-drugs may be comprised of structures whereina acid group is masked, for example, as an ester or amide. Furtherexamples of pro-drugs are discussed herein and, for example, byAlexander et al., J. Med. Chem. 1988, 31, 318 (hereby incorporated byreference in its entirety).

The term “pharmaceutically acceptable salt” is intended to include saltsderived from inorganic or organic acids including, for examplehydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric,formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic,salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic,trifluroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids;and salts derived from inorganic or organic bases including, for examplesodium, potassium, calcium, ammonium or tetrafluoroborate. Exemplarypharmaceutically acceptable salts are found, for example, in Berge, etal. (J. Pharm. Sci. 1977, 66(1), 1; and U.S. Pat. Nos. 6,570,013 and4,939,140; each hereby incorporated by reference in its entirety).Pharmaceutically acceptable salts are also intended to encompasshemi-salts, wherein the ratio of compound:acid is respectively 2:1.Exemplary hemi-salts are those salts derived from acids comprising twocarboxylic acid groups, such as malic acid, fumaric acid, maleic acid,succinic acid, tartaric acid, glutaric acid, oxalic acid, adipic acidand citric acid. Other exemplary hemi-salts are those salts derived fromdiprotic mineral acids such as sulfuric acid. Exemplary preferredhemi-salts include, but are not limited to, hemimaleate, hemifumarate,and hemisuccinate.

The term “acid” contemplates all pharmaceutically acceptable inorganicor organic acids. Inorganic acids include mineral acids such ashydrohalic acids, such as hydrobromic and hydrochloric acids, sulfuricacids, phosphoric acids and nitric acids. Organic acids include allpharmaceutically acceptable aliphatic, alicyclic and aromatic carboxylicacids, dicarboxylic acids, tricarboxylic acids, and fatty acids.Preferred acids are straight chain or branched, saturated or unsaturatedC1-C20 aliphatic carboxylic acids, which are optionally substituted byhalogen or by hydroxyl groups, or C6-C12 aromatic carboxylic acids.Examples of such acids are carbonic acid, formic acid, fumaric acid,acetic acid, propionic acid, isopropionic acid, valeric acid,alpha-hydroxy acids, such as glycolic acid and lactic acid, chloroaceticacid, benzoic acid, methane sulfonic acid, and salicylic acid. Examplesof dicarboxylic acids include oxalic acid, malic acid, succinic acid,tataric acid and maleic acid. An example of a tricarboxylic acid iscitric acid. Fatty acids include all pharmaceutically acceptablesaturated or unsaturated aliphatic or aromatic carboxylic acids having 4to 24 carbon atoms. Examples include butyric acid, isobutyric acid,sec-butyric acid, lauric acid, palmitic acid, stearic acid, oleic acid,linoleic acid, linolenic acid, and phenylsteric acid. Other acidsinclude gluconic acid, glycoheptonic acid and lactobionic acid.

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

An “effective amount”, “sufficient amount” or “therapeutically effectiveamount” as used herein is an amount of a compound that is sufficient toeffect beneficial or desired results, including clinical results. Assuch, the effective amount may be sufficient, for example, to reduce orameliorate the severity and/or duration of afflictions related to aldosereductase, or one or more symptoms thereof, prevent the advancement ofconditions or symptoms related to afflictions related to aldosereductase, or enhance or otherwise improve the prophylactic ortherapeutic effect(s) of another therapy. An effective amount alsoincludes the amount of the compound that avoids or substantiallyattenuates undesirable side effects.

As used herein and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results may include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminution of extent of disease or affliction, a stabilized(i.e., not worsening) state of disease or affliction, preventing spreadof disease or affliction, delay or slowing of disease or afflictionprogression, amelioration or palliation of the disease or afflictionstate and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The phrase “in need thereof” refers to the need for symptomatic orasymptomatic relief from conditions related to aldose reductase activityor that may otherwise be relieved by the compounds and/or compositionsof the invention.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Non-limiting examples of suchpharmaceutical carriers include liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical carriers may also be saline, gum acacia, gelatin,starch paste, talc, keratin, colloidal silica, urea, and the like. Inaddition, auxiliary, stabilizing, thickening, lubricating and coloringagents may be used. Other examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences (Alfonso Gennaroed., Krieger Publishing Company (1997); Remington's: The Science andPractice of Pharmacy, 21^(st) Ed. (Lippincot, Williams & Wilkins (2005);Modern Pharmaceutics, vol. 121 (Gilbert Banker and Christopher Rhodes,CRC Press (2002); each of which hereby incorporated by reference in itsentirety).

The terms “animal,” “subject” and “patient” as used herein include allmembers of the animal kingdom including, but not limited to, mammals(e.g., mice, rats, cats, monkeys, dogs, horses, swine, etc.) and humans.

In one embodiment, aldose reductase inhibitors described hereinencompass compounds of formula (I) or pharmaceutically acceptable salts,pro-drugs and solvates thereof,

wherein,

R¹ is H, (C₁-C₆)-alkyl, (C₁-C₆)-hydroxyalkyl, or (C₁-C₆)-aminoalkyl;

X¹ is N or CR³;

X² is N or CR⁴;

X³ is N or CR⁵;

X⁴ is N or CR⁶; with the proviso that two or three of X², X³, or X⁴ areN;

Y is a bond, C═O, C═S, C═NH, or C═N(C₁-C₄)-alkyl;

Z is

A¹ is NR¹¹, O, S or CH₂;

A² is N or CH;

A³ is NR¹¹, O, or S;

R³ through R¹⁰ are independently hydrogen, halogen, cyano, acyl,haloalkyl, haloalkoxy, haloalkylthio, trifluoroacetyl, (C₁-C₄)-alkyl,(C₁-C₄)-alkoxy, (C₁-C₄)-alkylthio, (C₁-C₄)-alkylsulfinyl, or(C₁-C₄)-alkylsulfonyl; or two of R³ through R⁶ or two of R⁷ through R¹⁰taken together are (C₁-C₄)-alkylenedioxy; and

R¹¹ is hydrogen, C1-C4 alkyl, or C(O)O—(C₁-C₄)-alkyl.

It will be recognized by those of skill in the art that the designationof

Z is

indicates that when Z is

the compounds of formula (I) are understood to encompass

and when Z is

the compounds of formula (I) are understood to encompass

In certain embodiments, R¹ is hydrogen or (C₁-C₆)-alkyl. In certainembodiments, R¹ is hydrogen. In certain embodiments, R¹ is(C₁-C₆)-alkyl. In certain embodiments, R¹ is tert-butyl.

In certain embodiments, R³ through R¹⁰ are independently hydrogen,halogen or haloalkyl. In certain embodiments, R³ through R¹⁰ areindependently hydrogen, halogen or trihaloalkyl.

In certain embodiments, R³ through R⁶ are hydrogen.

In certain embodiments, R⁷ through R¹⁰ are independently hydrogen,halogen or haloalkyl. In certain embodiments, R⁷ through R¹⁰ areindependently hydrogen, halogen or trihaloalkyl.

In certain embodiments, R⁷ and R¹⁰ are hydrogen.

In certain embodiments, R⁸ is hydrogen, halogen or haloalkyl. In certainembodiments, R⁸ is hydrogen. In certain embodiments, R⁸ is halogen. Incertain embodiments, R⁸ is haloalkyl.

In certain embodiments, R⁹ is hydrogen, halogen or haloalkyl. In certainembodiments, R⁹ is hydrogen. In certain embodiments, R⁹ is halogen. Incertain embodiments, R⁹ is haloalkyl.

In certain embodiments, Y is C═O, C═S, C═NH, or C═N(C₁-C₄)-alkyl. Incertain embodiments, Y is C═O or C═S. In certain embodiments, Y is C═O.In certain embodiments, Y is C═S. In certain embodiments, Y is C═NH, orC═N(C₁-C₄)-alkyl.

In certain embodiments, A¹ is NR¹¹, S or CH₂. In certain embodiments, A¹is NR¹¹ or O. In certain embodiments, A¹ is NR¹¹ or S. In certainembodiments, A¹ is NR¹¹. In certain embodiments, A¹ is O. In certainembodiments, A¹ is S.

In certain embodiments, A² is N or CH. In certain embodiments, A¹ is N.In certain embodiments, A¹ is CH.

In certain embodiments, A³ is O or S. In certain embodiments, A³ is O.In certain embodiments, A³ is S.

In certain embodiments, X¹ and X⁴ are nitrogen.

In certain embodiments, X¹ and X² are nitrogen.

In certain embodiments, X¹ and X³ are nitrogen.

In certain embodiments, X² and X³ are nitrogen.

In certain embodiments, X² and X⁴ are nitrogen.

In certain embodiments, X³ and X⁴ are nitrogen.

In certain embodiments, Z is

In certain embodiments, Z is

In certain embodiments, R¹ is hydrogen or (C₁-C₆)-alkyl;

X¹ and X⁴ are N;

X² is CR⁴;

X³ is CR⁵;

Y is C═O;

Z is

A¹ is NR¹¹, O, or S;

A² is N;

A³ is O, or S;

R⁴ and R⁵ are hydrogen;

R⁷ through R¹⁰ are independently hydrogen, halogen, cyano, acyl,haloalkyl, haloalkoxy, haloalkylthio, (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy,(C₁-C₄)-alkylthio, (C₁-C₄)-alkylsulfinyl, or (C₁-C₄)-alkylsulfonyl; and

R¹¹ is hydrogen, C₁-C₄ alkyl, or C(O)O—(C₁-C₄)-alkyl.

In certain embodiments, R¹ is hydrogen or tert-butyl;

X¹ and X⁴ are N;

X² is CR⁴;

X³ is CR⁵;

Y is C═O;

Z is

A¹ is NR¹¹, O or S;

A² is N;

A³ is O or S;

R⁴ and R⁵ are hydrogen;

R⁷ through R¹⁰ are independently hydrogen, halogen, or haloalkyl; and

R¹¹ is hydrogen, (C₁-C₄)-alkyl, or C(O)O-tert-butyl.

In certain embodiments, R¹ is hydrogen or tert-butyl;

X¹ and X⁴ are N;

X² is CH;

X³ is CH;

Y is C═O;

Z is

A¹ is NR¹¹, O or S;

A² is N;

A³ is O or S;

R⁷, R⁸ and R¹⁰ are independently hydrogen, halogen, or haloalkyl;

R⁹ is halogen, or haloalkyl; and

R¹¹ is hydrogen or methyl.

In certain embodiments, R¹ is hydrogen or tert-butyl;

X¹ and X⁴ are N;

X² is CH;

X³ is CH;

Y is C═O;

Z is

A¹ is NR¹¹, O or S;

A² is N;

A³ is O or S;

R⁷, R⁸ and R¹⁰ are independently hydrogen, halogen, or haloalkyl;

R⁹ is chlorine, or trifluromethyl; and

R¹¹ is hydrogen or methyl.

In certain embodiments, the compounds of formula (I) encompass CompoundA or pharmaceutically acceptable salts thereof, such as mono-, di-, ortri-ethanolamine salts.

In certain embodiments, the compounds of formula (I) encompass CompoundB or pharmaceutically acceptable salts thereof, such as mono-, di-, ortri-ethanolamine salts.

Synthesis

The compounds of formula (I) can generally be prepared, for example,according to Scheme 1.

where X¹, X², X³, X⁴, R¹, A¹, A², R³ through R¹¹ are defined as aboveand Q is a halogen, such as Cl, Br, I, and the like, or any otherleaving group, such as OH, OSO₂Me, OMs, OTs, OTf, and the like.

In certain embodiments, the reaction can be carried out in the presenceof a base, such as potassium tert-butoxide, sodium hydride, sodiummethoxide, sodium ethoxide, and the like.

In certain embodiments, the reaction can be carried out using aproticsolvents, such as DMF, THF, NMP, and the like. In certain embodiments,the reaction can be carried out using alcohol solvents, such asmethanol, ethanol, and the like.

In certain embodiments, the reaction can be carried out at temperaturesof between about 5° C. to about 80° C., such as 20° C. to 30° C.

In certain embodiments, the reaction can be subsequently followed byfurther separation and purification steps, such as chromatography (e.g.,flash, HPLC, MPLC, etc.), crystallization, and the like.

Other suitable reactions are possible, such as hydrolysis of thecompound of formula (I) in to obtain different forms of the compound offormula (I). For example, compounds having tert-butoxy, methoxy, ethoxy,and the like group as R¹ can be hydrolyzed by reacting with a suitablereagent, such as trifluoroacetic acid (TFA), HCl, KOH, or the like, toobtain a compound of formula (I) having hydrogen as R¹.

The compounds of formula (I) can also generally be prepared according toScheme 2.

For example, the following exemplary synthesis can be carried outaccording to Scheme 3.

In some other embodiments, where Y is C═O, subsequent reactions can becarried out to replace C═O with C═S or C═N, or the like.

Compound of Formula (IB)

To obtain compounds of Formula (IB), different possibilities exist. Forexample, commercial sources, such as Sigma-Aldrich may be available.Alternatively, compounds of Formula (IB) can be synthesized by a varietyof different reactions, such as a condensation reaction as schematicallyillustrated below in Scheme 4. The reaction can be carried out using avariety of solvents, such as ethanol, methanol, DMF, AcOH, and the like.The reaction can be carried out at temperatures of between about 5° C.to about 80° C., such as, for example, 55° C. to 65° C.

Additional exemplary descriptions regarding synthesis of certaincompounds of Formula (TB) are described in J. Med. Chem. (1991), Vol.34, pp. 108-122 and J. Med. Chem. (1992), Vol. 35, No. 3, pp. 457-465;each of which hereby incorporated by reference in its entirety.

Compounds of Formula (IA)

To obtain compounds of Formula (IA), different possibilities exist. Forexample, compounds of Formula (IA) can be synthesized as shown in Scheme5. For example, to obtain a compound of Formula (IA) when Y is C═O,reaction of a compound represented by Formula (IIA) with a reagent thatcause addition-cyclization reaction, such reaction with hydrazine or thelike, can be carried out as shown below. The reaction can be carried outusing a variety of solvents, such as ethanol, methanol, THF, and thelike. The reaction can be carried out at temperatures of between about20° C. to about 100° C., such as 60° C. to 80° C.

The compounds of Formula (IIA) can be obtained, for example, by areaction of an anhydride with a reagent that causes a Wittig reaction,such as (tert-butoxycarbonylmethylene)-triphenylphosphorane, and thelike, as shown in Scheme 6. The reaction can be carried out usingaprotic solvents, such as CH₂Cl₂, THF, 1,4-dioxane, toluene, and thelike. The reaction can be carried out at temperatures of between about20° C. to about 110° C., such as 55° C. to 70° C.

In certain embodiments, reaction of an anhydride with a reagent thatcauses a Wittig reaction can lead to a mixture of the particularcompounds represented by Formula (IIA), as exemplified below (Scheme 7).In such instances, if necessary, the mixture can be separated andpurified to obtain the particular compounds of Formula (IIA) ofinterest.

The compounds of Formula (IIIA) can generally be obtained throughcommercial sources, such as Sigma-Aldrich. Alternatively, compounds ofFormula (IIIA) can be obtained reaction of dicarboxylic acid derivativerepresented by Formula (IVA) with a suitable anhydride forming reagent,such as dicyclohexylcarbodiimide (DCC) or acetic anhydride, to obtainthe compounds of Formula (IIIA) as schematically illustrated below(Scheme 8). The reaction can be carried out using non-nucleophilicsolvents, such as acetic anhydride, THF, and the like. The reaction canbe carried out at temperatures of between about 20° C. to about 100° C.,such as 60° C. to 80° C.

The compounds of Formula (IVA) can generally be obtained throughcommercial sources, such as Sigma-Aldrich. Alternatively, compounds ofFormula (IVA) can be obtained reaction of suitable precursor representedby Formula (VA) with a suitable dicarboxylic acid derivative formingreagent, such as NaMnO₄ and/or NaOH, to obtain the compounds of Formula(IVA) as schematically illustrated below (Scheme 9). The reaction can becarried out using aqueous solvents, such as water. The reaction can becarried out at temperatures of between about 50° C. to about 100° C.,such as 85° C. to 95° C.

Additional Synthetic Schemes for Compound of Formula (I)

Additional reactions can be carried out for the synthesis of additionalembodiments of compounds represented by formula (I).

To obtain compounds of formula (I) where Y is C═S, the followingsynthesis can be carried out (Scheme 10).

To obtain compounds of Formula (I) where Y is C═NR*, wherein R*represents hydrogen or an alkyl substituent for example, the followingsynthesis can be carried out (Scheme 11).

Alternative reaction schemes may be possible. For example, the followingsynthetic scheme may be carried out to obtain compounds of formula (I)where Y is a covalent bond (Scheme 12).

In certain other embodiments, other types of reactions, such as thePerkins reaction, can be carried out to obtain compounds of formula (I)(Scheme 13). The Perkins reaction is shown illustrated below employingKOAc/Ac₂O. However, other temperatures and other bases, such as K₂CO₃and the like can be utilized.

Other substitutions and modifications are further possible as would beapparent to one of ordinary skill in the art. For example, in Scheme 13,KOH can be utilized in place of NaOH. In Scheme 14 below, KOtBu can beused in place of NaH. Additionally, instead of DMF, NMP or THF can beutilized. Additional details of the Perkins reaction can be found in WO03/061660, the contents of which are incorporated by reference herein inits entirety.

In certain embodiments, the following alternative synthesis can becarried out (Scheme 15).

Exemplary Reaction Schemes To Produce Embodiments Of Compounds OfFormula (I)

Particular, non-limiting, illustrative synthetic schemes for certainembodiments are shown as follows.

Compounds of formula (I) where three of X¹, X², X³, and X⁴ are nitrogenand Y is C═O may be synthesized, for example, according to generalScheme 16.

Compounds of formula (I) where X¹ and X⁴ are nitrogen and Y is C═O maybe synthesized, for example, according to general Scheme 17.

Compounds of formula (I) where X² and X³ are nitrogen and Y is C═O maybe synthesized, for example, according to general Scheme 18.

Compounds of formula (I) where X¹ and X² or X³ and X⁴ are nitrogen and Yis C═O may be synthesized, for example, according to general Scheme 19.

Compounds of formula (I) where X¹ and X³ or X² and X⁴ are nitrogen and Yis C═O may be synthesized, for example, according to general Scheme 20.

In these examples described above, Q is a halogen or a leaving group,and Ri and R2 are independently hydrogen, halogen (such as Cl or F) orhaloalkyl (such as CF3).

Compounds of formula (I) where X¹ and X⁴ are nitrogen and Y is acovalent bond may be synthesized, for example, according to generalScheme 21.

In the examples described above, the substituents are as describedpreviously herein.

Compounds or compositions of the invention can be useful in applicationsthat benefit from inhibition of aldose reductase enzymes. Exemplaryutility of aldose reductase inhibition may be found, for example, inU.S. Pat. No. 5,677,342; 5,155,259; 4,939,140; U.S. patent applicationSer. No. 11/210,283; and Roy et al., in Diabetes Research and ClinicalPractice, Vol. 10, Issue 1, 91-97; and references cited therein; each ofwhich hereby incorporated by reference in its entirety. Inhibition ofaldose reductase also has been found to prevent metastasis of coloncancer and mitosis in colon cancer cells (See, for example, Tammali, R.et al., Inhibition of Aldose Reductase Prevents Colon Cancer Metastasis,Carcinogenesis 2011, doi: 10.1093/carcin/bgr102; published online: Jun.3, 2011; Angiogenesis 2011 May;14(2):209-21; and Mol. Cancer Ther. 2010,April; 9(4): 813-824; each of which hereby incorporated by reference inits entirety).

In certain embodiments, compounds and/or compositions of the inventioncan be useful in promoting healthy aging of skin, the treatment of skindisorders, the treatment of angiogenesis disorders such as cancers,including colon cancer, the treatment of non-cardiac tissue damage, thetreatment of cardiovascular disorders, the treatment of renal disorders,the treatment of evolving myocardial infarction, and the treatmentvarious other disorders, such as complications arising from diabetes.Such disorders can include, but are not limited to, atherosclerosis,coronary artery disease, diabetic nephropathy, diabetic neuropathy,diabetic retinopathy, infections of the skin, peripheral vasculardisease, stroke, and the like.

In certain embodiments, compounds and/or compositions of the inventioncan be useful in cardiovascular applications. For example, compoundsand/or compositions of the invention can be used to treat patientsundergoing a heart bypass surgery to improve recovery after the surgery.In another example, compounds and/or compositions of the invention canbe used to inhibit or reduce accumulation or rapid onset ofatherosclerotic plaque.

In some other embodiments, compounds and/or compositions of theinvention can be useful in topical applications. For example, compoundsand/or compositions of the invention can be used to retard or reduceskin aging.

In certain embodiments, compounds of formula (I) can be administered toa subject in need of treatment at dosages ranging from about 0.5 toabout 25 mg/kg body weight of the subject to be treated per day, such asfrom about 1.0 to 10 mg/kg. However, additional variations are withinthe scope of the invention.

The compound of formula (I) can be administered alone or in combinationwith pharmaceutically acceptable carriers, such as diluents, fillers,aqueous solution, and even organic solvents. The compound and/orcompositions of the invention can be administered as a tablet, powder,lozenge, syrup, injectable solution, and the like. Additionalingredients, such as flavoring, binder, excipients, and the like arewithin the scope of the invention.

In certain embodiments, pharmaceutically acceptable compositions cancontain a compound of formula (I) and/or a pharmaceutically acceptablesalt thereof at a concentration ranging from about 0.01 to about 2 wt%,such as 0.01 to about 1 wt % or about 0.05 to about 0.5 wt %. Thecomposition can be formulated as a solution, suspension, ointment, or acapsule, and the like. The pharmaceutical composition can be prepared asan aqueous solution and can contain additional components, such aspreservatives, buffers, tonicity agents, antioxidants, stabilizers,viscosity-modifying ingredients and the like.

Other equivalent modes of administration can be found in U.S. Pat. No.4,939,140, hereby incorporated by reference herein in its entirety.

In one embodiment, the present invention provides for the use ofpharmaceutical compositions and/or medicaments comprised of a compoundof formula I, or a pharmaceutically acceptable salt, hydrate, solvate,or pro-drug thereof, in a method of treating a disease state, and/orcondition caused by or related to aldose reductase.

In another embodiment, the method of treatment comprises the steps of:i) identifying a subject in need of such treatment; (ii) providing acompound of formula I, or a pharmaceutically acceptable salt, hydrate,solvate, pro-drug or tautomer thereof; and (iii) administering saidcompound of formula I in a therapeutically effective amount to treat,suppress and/or prevent the disease state or condition in a subject inneed of such treatment.

In another embodiment, the method of treatment comprises the steps of:i) identifying a subject in need of such treatment; (ii) providing acomposition comprising a compound of formula I, or a pharmaceuticallyacceptable salt, hydrate, solvate, pro-drug or tautomer thereof; and(iii) administering said composition in a therapeutically effectiveamount to treat, suppress and/or prevent the disease state or conditionin a subject in need of such treatment.

In one embodiment, the compound or composition is administered orally.

In one embodiment, the methods comprise administering to the subject aneffective amount of a compound of formula I, or a pharmaceuticallyacceptable salt, solvate, hydrate or pro-drug thereof; or a compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt, solvate, hydrate or pro-drug thereof, and a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carriers arewell-known to those skilled in the art, and include, for example,adjuvants, diluents, excipients, fillers, lubricants and vehicles.Often, the pharmaceutically acceptable carrier is chemically inerttoward the active compounds and is non-toxic under the conditions ofuse. Examples of pharmaceutically acceptable carriers may include, forexample, water or saline solution, polymers such as polyethylene glycol,carbohydrates and derivatives thereof, oils, fatty acids, or alcohols.

In another embodiment, the method of treatment, prevention and/orsuppression of a condition related to aldose reductase comprises thesteps of: i) identifying a subject in need of such treatment; (ii)providing a compound of formula I, or a pharmaceutically acceptablesalt, solvate, hydrate or pro-drug thereof or a composition comprising acompound of formula I, or a pharmaceutically acceptable salt, solvate,hydrate or pro-drug thereof, and a pharmaceutically acceptable carrier;and (iii) administering said compound or composition in atherapeutically effective amount to treat, prevent and/or suppress thedisease state or condition related to aldose reductase in a subject inneed of such treatment.

In one embodiment, the present invention also encompasses methodscomprising pro-drugs of compounds of formula I and/or pharmaceuticalcompositions thereof. Pro-drugs include derivatives of compounds thatcan hydrolyze, oxidize, or otherwise react under biological conditions(in vitro or in vivo) to provide an active compound of the invention.Examples of pro-drugs include, but are not limited to, derivatives andmetabolites of a compound of the invention that include biohydrolyzablemoieties such as biohydrolyzable amides, biohydrolyzable esters,biohydrolyzable carbamates, biohydrolyzable carbonates, andbiohydrolyzable phosphate analogues. Pro-drugs are also described in,for example, The Practice of Medicinal Chemistry (Camille Wermuth, ed.,1999, Academic Press; hereby incorporated by reference in its entirety).In certain embodiments, pro-drugs of compounds with carboxyl functionalgroups are the lower alkyl esters of the carboxylic acid. Thecarboxylate esters are conveniently formed by esterifying any of thecarboxylic acid moieties present on the molecule. Pro-drugs cantypically be prepared using well-known methods, such as those describedby Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J.Abraham ed., 2001, Wiley) and Design and Application of Pro-drugs (H.Bundgaard ed., 1985, Harwood Academic Publishers Gmfh; each of whichhereby incorporated by reference in its entirety). Biohydrolyzablemoieties of a compound of formula I 1) do not interfere with thebiological activity of the compound but can confer upon that compoundadvantageous properties in vivo, such as uptake, duration of action, oronset of action; or 2) may be biologically inactive but are converted invivo to the biologically active compound. Examples of biohydrolyzableesters include, but are not limited to, lower alkyl esters,alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, a-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

In one embodiment, the compounds of the invention are formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo.According to another aspect, the present invention provides apharmaceutical composition comprising a compound of formula I inadmixture with a pharmaceutically acceptable diluent and/or carrier. Thepharmaceutically-acceptable carrier is “acceptable” in the sense ofbeing compatible with the other ingredients of the composition and notdeleterious to the recipient thereof. The pharmaceutically-acceptablecarriers employed herein may be selected from various organic orinorganic materials that are used as materials for pharmaceuticalformulations and which are incorporated as analgesic agents, buffers,binders, disintegrants, diluents, emulsifiers, excipients, extenders,glidants, solubilizers, stabilizers, suspending agents, tonicity agents,vehicles and viscosity-increasing agents. Pharmaceutical additives, suchas antioxidants, aromatics, colorants, flavor-improving agents,preservatives, and sweeteners, may also be added. Examples of acceptablepharmaceutical carriers include carboxymethyl cellulose, crystallinecellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc andwater, among others. In one embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

Surfactants such as, for example, detergents, are also suitable for usein the formulations. Specific examples of surfactants includepolyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetateand of vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol,glycerol, sorbitol or polyoxyethylenated esters of sorbitan; lecithin orsodium carboxymethylcellulose; or acrylic derivatives, such asmethacrylates and others, anionic surfactants, such as alkalinestearates, in particular sodium, potassium or ammonium stearate; calciumstearate or triethanolamine stearate; alkyl sulfates, in particularsodium lauryl sufate and sodium cetyl sulfate; sodiumdodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fattyacids, in particular those derived from coconut oil, cationicsurfactants, such as water-soluble quaternary ammonium salts of formulaN⁺R′R″R′″R″″Y⁻, in which the R radicals are identical or differentoptionally hydroxylated hydrocarbon radicals and Y⁻ is an anion of astrong acid, such as halide, sulfate and sulfonate anions;cetyltrimethylammonium bromide is one of the cationic surfactants whichcan be used, amine salts of formula N⁺R′R″R′″, in which the R radicalsare identical or different optionally hydroxylated hydrocarbon radicals;octadecylamine hydrochloride is one of the cationic surfactants whichcan be used, non-ionic surfactants, such as optionallypolyoxyethylenated esters of sorbitan, in particular Polysorbate 80, orpolyoxyethylenated alkyl ethers; polyethylene glycol stearate,polyoxyethylenated derivatives of castor oil, polyglycerol esters,polyoxyethylenated fatty alcohols, polyoxyethylenated fatty acids orcopolymers of ethylene oxide and of propylene oxide, amphotericsurfactants, such as substituted lauryl compounds of betaine,

When administered to a subject, the compound of formula I andpharmaceutically acceptable carriers can be sterile. Suitablepharmaceutical carriers may also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, polyethylene glycol 300,water, ethanol, polysorbate 20, and the like. The present compositions,if desired, may also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents.

The pharmaceutical formulations of the present invention are prepared bymethods well-known in the pharmaceutical arts. Optionally, one or moreaccessory ingredients (e.g., buffers, flavoring agents, surface activeagents, and the like) also are added. The choice of carrier isdetermined by the solubility and chemical nature of the compounds,chosen route of administration and standard pharmaceutical practice.

Additionally, the compounds and/or compositions of the present inventionare administered to a human or animal subject by known proceduresincluding oral administration, sublingual or buccal administration. Inone embodiment, the compound and/or composition is administered orally.

For oral administration, a formulation of the compounds of the inventionmay be presented in dosage forms such as capsules, tablets, powders,granules, or as a suspension or solution. Capsule formulations may begelatin, soft-gel or solid. Tablets and capsule formulations may furthercontain one or more adjuvants, binders, diluents, disintegrants,excipients, fillers, or lubricants, each of which are known in the art.Examples of such include carbohydrates such as lactose or sucrose,dibasic calcium phosphate anhydrous, corn starch, mannitol, xylitol,cellulose or derivatives thereof, microcrystalline cellulose, gelatin,stearates, silicon dioxide, talc, sodium starch glycolate, acacia,flavoring agents, preservatives, buffering agents, disintegrants, andcolorants. Orally administered compositions may contain one or moreoptional agents such as, for example, sweetening agents such asfructose, aspartame or saccharin; flavoring agents such as peppermint,oil of wintergreen, or cherry; coloring agents; and preservative agents,to provide a pharmaceutically palatable preparation.

In some embodiments, the composition is in unit dose form such as atablet, capsule or single-dose vial. Suitable unit doses, i.e.,therapeutically effective amounts, may be determined during clinicaltrials designed appropriately for each of the conditions for whichadministration of a chosen compound is indicated and will, of course,vary depending on the desired clinical endpoint.

In accordance with the methods of the present invention, the compoundsof the invention are administered to the subject in a therapeuticallyeffective amount, for example to reduce or ameliorate symptoms relatedto aldose reductase activity in the subject. This amount is readilydetermined by the skilled artisan, based upon known procedures,including analysis of titration curves established in vivo and methodsand assays disclosed herein.

In one embodiment, the methods comprise administration of atherapeutically effective dosage of the compounds of the invention. Insome embodiments, the therapeutically effective dosage is at least about0.05 mg/kg body weight, at least about 0.1 mg/kg body weight, at leastabout 0.25 mg/kg body weight, at least about 0.3 mg/kg body weight, atleast about 0.5 mg/kg body weight, at least about 0.75 mg/kg bodyweight, at least about 1 mg/kg body weight, at least about 2 mg/kg bodyweight, at least about 3 mg/kg body weight, at least about 4 mg/kg bodyweight, at least about 5 mg/kg body weight, at least about 6 mg/kg bodyweight, at least about 7 mg/kg body weight, at least about 8 mg/kg bodyweight, at least about 9 mg/kg body weight, at least about 10 mg/kg bodyweight, at least about 15 mg/kg body weight, at least about 20 mg/kgbody weight, at least about 25 mg/kg body weight, at least about 30mg/kg body weight, at least about 40 mg/kg body weight, at least about50 mg/kg body weight, at least about 75 mg/kg body weight, at leastabout 100 mg/kg body weight, at least about 200 mg/kg body weight, atleast about 250 mg/kg body weight, at least about 300 mg/kg body weight,at least about 350 mg/kg body weight, at least about 400 mg/kg bodyweight, at least about 450 mg/kg body weight, at least about 500 mg/kgbody weight, at least about 550 mg/kg body weight, at least about 600mg/kg body weight, at least about 650 mg/kg body weight, at least about700 mg/kg body weight, at least about 750 mg/kg body weight, at leastabout 800 mg/kg body weight, at least about 900 mg/kg body weight, or atleast about 1000 mg/kg body weight. It will be recognized that any ofthe dosages listed herein may constitute an upper or lower dosage range,and may be combined with any other dosage to constitute a dosage rangecomprising an upper and lower limit.

In some embodiments, the methods comprise a single dosage oradministration (e.g., as a single injection or deposition).Alternatively, the methods comprise administration once daily, twicedaily, three times daily or four times daily to a subject in needthereof for a period of from about 2 to about 28 days, or from about 7to about 10 days, or from about 7 to about 15 days, or longer. In someembodiments, the methods comprise chronic administration. In yet otherembodiments, the methods comprise administration over the course ofseveral weeks, months, years or decades. In still other embodiments, themethods comprise administration over the course of several weeks. Instill other embodiments, the methods comprise administration over thecourse of several months. In still other embodiments, the methodscomprise administration over the course of several years. In still otherembodiments, the methods comprise administration over the course ofseveral decades.

The dosage administered can vary depending upon known factors such asthe pharmacodynamic characteristics of the active ingredient and itsmode and route of administration; time of administration of activeingredient; age, sex, health and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency of treatmentand the effect desired; and rate of excretion. These are all readilydetermined and may be used by the skilled artisan to adjust or titratedosages and/or dosing regimens.

The precise dose to be employed in the compositions will also depend onthe route of administration, and should be decided according to thejudgment of the practitioner and each patient's circumstances. Inspecific embodiments of the invention, suitable dose ranges for oraladministration of the compounds of the invention are generally about 1mg/day to about 1000 mg/day. In one embodiment, the oral dose is about 1mg/day to about 800 mg/day. In one embodiment, the oral dose is about 1mg/day to about 500 mg/day. In another embodiment, the oral dose isabout 1 mg/day to about 250 mg/day. In another embodiment, the oral doseis about 1 mg/day to about 100 mg/day. In another embodiment, the oraldose is about 5 mg/day to about 50 mg/day. In another embodiment, theoral dose is about 5 mg/day. In another embodiment, the oral dose isabout 10 mg/day. In another embodiment, the oral dose is about 20mg/day. In another embodiment, the oral dose is about 30 mg/day. Inanother embodiment, the oral dose is about 40 mg/day. In anotherembodiment, the oral dose is about 50 mg/day. In another embodiment, theoral dose is about 60 mg/day. In another embodiment, the oral dose isabout 70 mg/day. In another embodiment, the oral dose is about 100mg/day. It will be recognized that any of the dosages listed herein mayconstitute an upper or lower dosage range, and may be combined with anyother dosage to constitute a dosage range comprising an upper and lowerlimit.

Any of the compounds and/or compositions of the invention may beprovided in a kit comprising the compounds and/or compositions. Thus, inone embodiment, the compound and/or composition of the invention isprovided in a kit.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be within the scope of the present invention.

The invention is further described by the following non-limitingExamples.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples serve to illustrate theexemplary modes of making and practicing the invention. However, thescope of the invention is not to be construed as limited to specificembodiments disclosed in these Examples, which are illustrative only.

Example 1 Preparation of Compound A

Compound A was prepared as schematically illustrated below.

Preparation of (E)/(Z)-tent-butyl2-(7-oxofuro[3,4-b]pyrazin-5(7H)-ylidene) acetate (Compound 1)

To a stirred solution of 5.05 g (33.63 mmol) of commercially available2,3-pyrazinedicarboxylic anhydride in 300 mL of CHC13 was added 12.34 g(33.63 mmol) of (tert-butoxycarbonylmethylene)-triphenylphosphorane. Theresulting solution was heated to 62° C. for 2 days. The reaction mixturewas concentrated in vacuo and the residue purified via flash columnchromatography over silica gel (monitored by thin layer chromatography)and eluted with 1:1 (v/v) hexanes:ethyl acetate. Evaporation of thecollected fractions yielded 2.99 g (36% yield) of (E)/(Z)-tent-butyl2-(7-oxofuro[3,4-b]pyrazin-5(7H)-ylidene)acetate (Compound 1) as amixture of geometrical isomers (˜1:1) that was not separated: ¹H NMR(CDCl₃, 300 MHz): δ_(ppm) 9.03 (d, J=2.4 Hz, 1H), 8.96 (d, J=2.4 Hz,1H), 8.92 (d, J=2.4 Hz, 1H), 8.90 (d, J=2.4 Hz, 1H), 6.32 (s, 2H), 1.58(s, 18H).

Preparation of tert-butyl2-(8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl) acetate (Compound 2)

To a stirred solution of 9.42 g (37.99 mmol) of (E)/(Z)-tert-butyl2-(7-oxofuro [3,4-b]pyrazin-5(7H)-ylidene)acetate (Compound 1) in 600 mLof ethanol was added 1.25 mL (39.90 mmol) of hydrazine. The resultingsolution was brought to 80° C. for 3 hours. Subsequently, the reactionmixture was concentrated in vacuo and the residue purified via flashcolumn chromatography over silica gel (monitored by thin layerchromatography) and eluted with 19:1 (v/v) methylene chloride:methanol.Evaporation of the collected fractions yielded 7.78 g (78% yield) oftert-butyl 2-(8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl) acetate(Compound 2): ¹H NMR (CDCl₃, 300 MHz): δ_(ppm) 10.67 (br s, 1H), 9.06(d, J=2.1 Hz, 1H), 9.04 (d, J=2.1 Hz, 1H), 4.02 (s, 2H), 1.43 (s, 9H).

Preparation of tent-butyl2-(8-oxo-7-((5-(trifluoromethyl)benzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 4)

To a stirred solution of 7.78 g (29.58 mmol) of tent-butyl2-(8-oxo-7,8-dihydropyrazino [2,3-d]pyridazin-5-yl)acetate (Compound 2)in 300 mL of DMF was added 3.49 g (31.06 mmol) of potassiumtert-butoxide. The resulting reaction mixture was stirred at ambienttemperature for 0.5 hours. Subsequently, a solution of 7.80 g (31.06mmol) of 2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole (Compound3) in 20 mL of DMF was added and the resulting reaction mixture stirredat ambient temperature overnight. The reaction mixture was thenpartitioned between ethyl acetate and water, the layers separated, andthe ethyl acetate layer washed with a copious amount of water (3×). Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuo.The residue was purified via flash column chromatography over silica gel(monitored by thin layer chromatography) and eluted with a gradient of3:1 (v/v) hexanes:ethyl acetate to 1:1 (v/v) hexanes:ethyl acetate. Theobtained residue was then rechromatographed over silica gel and elutedwith 49:1 (v/v) methylene chloride:methanol. Evaporation of thecollected fractions yielded 6.88 g (48% yield) of tert-butyl2-(8-oxo-7-((5-(trifluoromethyl)benzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 4): tH NMR (CDCl₃, 300 MHz):δ_(ppm) 9.08 (d, J=2.1 Hz, 1H), 9.04 (d, J=2.1 Hz, 1H), 8.27 (s, 1H),7.94 (d, J=8.4 Hz, 1H), 7.61 (d, J=8.4 Hz, 1H), 5.89 (s, 2H), 4.04 (s,2H), 1.42 (s, 9H).

Preparation of2-(8-oxo-7-((5-(trifluoromethyl)benzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound A)

To a stirred solution of 6.0 g (12.55 mmol) of tent-butyl2-(8-oxo-7-((5-(trifluoromethyl)benzo-[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate(Compound 4) in 41 mL of CH₂Cl₂ was added 82 mL of TFA. The resultingreaction mixture was stirred at ambient temperature for 1 hour.Subsequently, the reaction mixture was concentrated in vacuo and theresidue partitioned between ethyl acetate and 1.0 M KOH in water. Thelayers were separated and the aqueous layer extracted with ethyl acetate(2×). The aqueous layer was acidified to a pH ˜2 with concentrated HCland subsequently extracted with ethyl acetate (3×). The organics fromthe second extraction were dried over Na₂SO₄, filtered and concentratedin vacuo. The residue was purified via flash column chromatography oversilica gel (monitored by thin layer chromatography) and eluted with 19:1(v/v) methylene chloride:methanol containing 1% (by volume) acetic acid.Evaporation of the collected fractions yielded 2.30 g (44% yield) of2-(8-oxo-7-((5-(trifluoromethyl)benzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound A) as a solid: ¹H NMR (DMSO-d₆, 300 MHz):6_(ppm)9.26 (d, J=2.1 Hz, 1H), 9.22 (d, J=2.1 Hz, 1H), 8.37 (s, 1H),8.35 (d, J=8.4 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H), 5.88 (s, 2H), 4.03 (s,2H); m.p.=192-193° C.

Example 2 Alternate Preparation of Compound A

Preparation of 2-(iodomethyl)-5-(trifluoromethyl)benzo[d]thiazole(Compound 5)

To a stirred solution of 10.79 g (42.97 mmol) of2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole (Compound 3) in 86mL of acetone was added 7.40 g (49.42 mmol) of sodium iodide. Theresulting reaction mixture was heated to 55° C. for 1 hour. The reactionmixture was cooled to ambient temperature and concentrated in vacuo. Theresidue was partitioned between EtOAc and water, the layers separated,and the organic layer washed with water (1×). The recovered organiclayer was then treated with 1.0 M Na₂S₂O₃ and stirred vigorously for 15minutes. The layers were then separated and the organic layer washedsequentially with water (1×) and brine (1×). The organic layer was driedover Na₂SO₄, filtered and concentrated in vacuo to yield 14.28 g (97%crude yield) of 2-(iodomethyl)-5-(trifluoromethyl)benzo[d]thiazole(Compound 5) that was used without further purification: ¹H NMR (CDCl₃,300 MHz): δ_(ppm) 8.26 (s, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.66 (d, J=8.4Hz, 1H), 4.80 (s, 2H).

Preparation of 3-(methoxycarbonyl)pyrazine-2-carboxylic acid (Compound6)

A solution of 12.0 g (79.95 mmol) of 2,3-pyrazine dicarboxylic anhydridein 282 mL of MeOH was heated to 65° C. overnight. The reaction mixturewas cooled to ambient temperature and concentrated in vacuo. To theobtained residue was added water followed by slow addition of solidNaHCO₃. After gas evolution ceased, the aqueous layer was extracted withEtOAc (1×). The aqueous layer was then acidified to pH 2 by addition ofconc. HCl. The aqueous layer was extracted with EtOAc (2×) and thecombined organics from the second extraction washed with brine (1×). Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuoto yield 12.41 g (85% crude yield) of 3-(methoxycarbonyl)pyrazine-2-carboxylic acid (Compound 6) as a white powder that was usedwithout further purification: ¹H NMR (DMSO, 300 MHz): δ_(ppm) 8.91 (d,J=2.7 Hz, 1H), 8.89 (d, J=2.7 Hz, 1H), 3.91 (s, 3H).

Preparation of tert-butyl2-(8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl) acetate (Compound 2)

In flask #1, a solution of 12.23 g (67.22 mmol) of 3-(methoxycarbonyl)pyrazine-2-carboxylic acid (Compound 6) in 88 mL of DMF was treatedslowly with 12.53 g (77.28 mmol) of CDI. The reaction mixture wasstirred at ambient temperature for 2 hours.

In a separate flask, flask #2, to 147 mL of DMF cooled to 0° C. wasadded portionwise 8.32 g (87.39 mmol) of MgCl₂. After stirring at 0° C.for 5 minutes, 13.5 mL (87.39 mmol) of mono-tent-butyl malonate and 37.4mL (269 mmol) of triethylamine was added and the resulting reactionmixture stirred at ambient temperature for 2 hours. After 2 hours, thecontent of flask #1 was added to flask #2 and the combined reactionmixture stirred at ambient temperature overnight. Subsequently, thereaction mixture was poured into aqueous 1.0 M HCl cooled to 0° C. andstirred for 15 minutes. To the mixture was added Et₂O, the layersseparated, and the ethereal layer washed sequentially with water (1×),saturated aqueous NaHCO3 (1×), water (1×) and brine (1×). The organicorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuoto yield crude methyl 3-(3-(tert-butoxy)-3-oxopropanoyl)pyrazine-2-carboxylate (Compound 7) that was used without furtherpurification.

The crude methyl3-(3-(tert-butoxy)-3-oxopropanoyl)pyrazine-2-carboxylate (Compound 7)was taken up in 250 mL of MeOH and the resulting solution cooled to 0°C. Subsequently, 2.2 mL (70.58 mmol) of hydrazine was added in adrop-wise fashion and the reaction mixture was warmed to ambienttemperature for 1 hour. After 1 hour, the reaction mixture wasconcentrated in vacuo and the residue purified via flash columnchromatography over silica gel (monitored by thin layer chromatography)and eluted with 19:1 (v/v) methylene chloride:methanol. Evaporation ofthe collected fractions yielded a yellow solid that was further purifiedvia recrystallization in EtOAc to yield 13.23 g (75% yield) oftert-butyl 2-(8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate(Compound 2): ¹H NMR (CDCl₃, 300 MHz): δ_(ppm) 10.3 (br s, 1H), 9.06 (d,J=2.1 Hz, 1H), 9.04 (d, J=2.1 Hz, 1H), 4.02 (s, 2H), 1.43 (s, 9H).

Preparation of tert-butyl2-(8-oxo-7-((5-(trifluoromethyObenzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 4)

To a vigorously stirred solution of 2.5 g (9.54 mmol) of tert-butyl2-(8-oxo-7,8-dihydropyrazino [2,3-d]pyridazin-5-yl)acetate (Compound 2)in 67 mL of NMP was added 3.12 g (9.09 mmol) of2-(iodomethyl)-5-(trifluoromethyObenzo[d]thiazole (Compound 5) and 1.51g (10.9 mmol) of K2CO3. The resulting reaction mixture was covered fromlight and stirred for 5 hours at ambient temperature. Subsequently, tothe reaction mixture was added Et2O and water, the layers separated, andthe aqueous layer extracted with Et2O (1×). The combined ethereal layerswere then washed sequentially with H₂O (1×), 1.0 M KOH (1×), 1.0 MNa₂S₂O₃ (1×), 1.0 M HCl (1×) and brine (1×). The organic layer was driedover Na₂SO₄, filtered and concentrated in vacuo. The residue waspurified via flash column chromatography over silica gel (monitored bythin layer chromatography) and eluted with 49:1 (v/v) methylenechloride:methanol. Evaporation of the collected fractions yielded 4.03 g(93% yield) of tert-butyl2-(8-oxo-7-((5-(trifluoromethyObenzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 4).

Preparation of2-(8-oxo-7-((5-(trifluoromethyObenzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound A)

(The deprotection of Compound 4 occurred in two separate flasks, butbefore workup and purification took place the two reaction mixtures werecombined together).

In one flask, 3.09 g (6.49 mmol) of tert-butyl2-(8-oxo-7-((5-(trifluoromethyl)benzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate(Compound 4) was dissolved in 30 mL of formic acid (88% in water) and3.0 mL of water. In a separate flask, 4.95 g (10.37 mmol) of Compound 4was dissolved in 48 mL of formic acid (88% in water) and 5.0 mL ofwater. The reaction mixtures were stirred separately for 22 hours atambient temperature. The reaction mixtures were concentrated in vacuoand the residues combined. The combined residues were partitionedbetween Et₂O and saturated aqueous NaHCO₃, the layers separated, and theaqueous layer extracted with Et₂O (1×). The aqueous layer was acidifiedto pH 2 by addition of conc. HCl and was extracted with EtOAc (3×). Theorganic layer from the second extraction was dried over Na₂SO₄, filteredand concentrated in vacuo. The residue was purified via flash columnchromatography over silica gel (monitored by thin layer chromatography)and eluted with 97:3 (v/v) methylene chloride:methanol containing 1%AcOH. Evaporation of the collected fractions yielded 5.29 g (75% yield)of 2-(8-oxo-7-((5-(trifluoromethyObenzo[d]thiazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (CompoundA) as a solid. The solid can be further purified via recrystallizationfrom MeOH to yield an off-white solid: m.p. =210-211° C. ¹H NMR(acetone-d6, 400 MHz): δ_(ppm) 9.19 (d, J=2.0Hz, 1H), 9.17 (d, J=2.0Hz,1H), 8.30-8.27 (m, 2H), 7.74 (dd, J=8Hz, 1.2Hz, 1H), 5.90 (s, 2H), 4.12(s, 2H); ESI-MS 422 (M+H)+; m.p.=210-211° C.

Example 3 Preparation of Compound 8

Compound 8, shown below, was prepared as follows:

The preparation described for Compound 4 was repeated except that5-chloro-2-(chloromethyl)-benzo [d]thiazole was the reagent employed inplace of 2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, usingthe same molar proportions as before. In this case, the final productobtained was tert-butyl2-(7-((5-chlorobenzo[d]thiazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 8) in 75% yield: ¹H NMR (CDCl₃,300 MHz): δ_(ppm) 9.08 (s, 1H), 9.05 (s, 1H), 8.00 (s, 1H), 7.74 (d,J=8.7 Hz, 1H), 7.36 (d, J=8.7 Hz, 1H), 5.87 (s, 2H), 4.04 (s, 2H), 1.41(s, 9H).

Example 4 Preparation of Compound B

Compound B, shown below, was prepared as follows:

The preparation described for Compound 8 was repeated. The schemesdescribed in Example 1 to obtain Compound A from Compound 4 was carriedout, where Compound 4 was replaced with Compound 8. In this case, thefinal product obtained was 2-(7-((5-chlorobenzo[d]thiazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound B) in 51% yield: : ¹H NMR (DMSO-d6, 300 MHz):δ_(ppm) 9.26 (d, J =2.1 Hz, 1H), 9.21 (d, J=2.1 Hz, 1H), 8.13 (d, J=8.7Hz, 1H), 8.09 (d, J=2.4 Hz, 1H), 7.51 (dd, J=8.7, 2.4 Hz, 1H), 5.83 (s,2H), 4.02 (s, 2H); m.p. =196-197° C.

Example 5 Preparation of Compound 9

The preparation described for Compound 4 was repeated except that2-(bromomethyl)-5-fluorobenzo [d]thiazole was the reagent employed inplace of 2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, usingthe same molar proportions as before. In this case, the final productobtained was tert-butyl 2-(7-((5-fluorobenzo[d]thiazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound9) in 73% yield: ¹H NMR (CDCl₃, 300 MHz): δ_(ppm) 9.09 (d, J=1.8 Hz,1H), 9.05 (d, J=1.8 Hz, 1H), 7.77 (dd, J=8.7 Hz, 4.8 Hz, 1H), 7.71 (dd,J=9.3 Hz, 2.7 Hz, 1H), 7.16 (ddd, J=8.7 Hz, 8.7Hz, 2.7 Hz, 1H), 5.88 (s,2H), 4.05 (s, 2H), 1.42 (s, 9H).

Example 6 Preparation of Compound 10.

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 9. In thiscase, the final product obtained was2-(7-((5-fluorobenzo[d]thiazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound 10) in 63% yield: ¹H NMR(DMSO-d6, 300 MHz): δ_(ppm) 9.25 (d, J=2.1 Hz, 1H), 9.21 (d, J=2.1 Hz,1H), 8.12 (dd, J=9.0 Hz, 5.7 Hz, 1H), 7.85 (dd, J=9.9 Hz, 2.4 Hz, 1H),7.36 (ddd, J=9.0 Hz, 9.0 Hz, 2.4 Hz, 1H), 5.82 (s, 2H), 4.02 (s, 2H).

Example 7 Preparation of Compound 11.

The preparation described for Compound 4 was repeated except that2-(bromomethyl) benzo[d]thiazole was the reagent employed in place of2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, using the samemolar proportions as before. In this case, the final product obtainedwas tert-butyl2-(7-(benzo[d]thiazol-2-ylmethyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 11) in 63% yield: ¹H NMR (CDCl₃,300 MHz): δ_(ppm) 9.07 (d, J=1.8 Hz, 1H), 9.03 (d, J=1.8 Hz, 1H), 8.02(d, J=7.5 Hz, 1H), 7.83 (d, J=7.5 Hz, 1H), 7.46 (ddd, J=7.5 Hz, 7.5 Hz,1.2 Hz, 1H), 7.37 (ddd, J=7.5 Hz, 7.5 Hz, 1.2 Hz, 1H), 5.89 (s, 2H),4.04 (s, 2H), 1.41 (s, 9H).

Example 8 Preparation of Compound 12.

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 11. In thiscase, the final product obtained was2-(7-(benzo[d]thiazol-2-ylmethyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)aceticacid (Compound 12) in 67% yield: : ¹H NMR (DMSO-d6, 300 MHz): δ_(ppm)9.26 (d, J=2.1 Hz, 1H), 9.22 (d, J=2.1 Hz, 1H), 8.08 (dd, J=7.5 Hz, 1.2Hz, 1H), 7.99 (dd, J=8.1 Hz, 1.2 Hz, 1H), 7.52 (ddd, J=8.1 Hz, 8.1 Hz,1.2 Hz, 1H), 7.45 (ddd, J=7.5 Hz, 7.5 Hz, 1.2 Hz, 1H), 5.83 (s, 2H),4.03 (s, 2H).

Example 9 Preparation of Compound 13.

The preparation described for Compound 4 was repeated except that2-(chloromethyl)-1-methyl-1H-benzo [d]imidazole was the reagent employedin place of 2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, usingthe same molar proportions as before. In this case, the final productobtained was tert-butyl 2-(7-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate(Compound 13) in 50% yield: tH NMR (CDCl₃, 300 MHz): δ_(ppm) 9.04 (d,J=2.1 Hz, 1H), 9.02 (d, J=2.1 Hz, 1H), 7.74 (dd, J=7.2 Hz, 1.2 Hz, 1H),7.35 (ddd , J=7.2 Hz, 7.2 Hz, 1.2 Hz, 1H), 7.29-7.22 (m, 2H), 5.77 (s,2H), 4.05 (s, 2H), 3.97 (s, 3H), 1.40 (s, 9H).

Example 10 Preparation of Compound 14.

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 13. In thiscase, the final product obtained was2-(7-1H-methyl-1H-benzo[d]imidazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound 14) in 88% yield: : ¹H NMR(DMSO-d_(6, 300) MHz): _(pp)m 9.26 (d, J=2.1 Hz, 1H), 9.21 (d, J=2.1 Hz,1H), 7.70 (d, J=8.4 Hz, 1H), 7.61 (d, J=8.1 Hz, 1H), 7.37 (dd, J=7.8 Hz,7.8 Hz, 1H), 7.29 (dd, J=7.8 Hz, 7.8 Hz, 1H), 5.81 (s, 2H), 4.00 (s,2H), 3.97 (s, 3H).

Example 11 Preparation of Compound 15.

To a solution of 0.100g (0.427 mmol) of2-(chloromethyl)-5-(trifluoromethyl)-1H-benzo [d]imidazole in 4.0 mL ofCH₂Cl₂ was added sequentially 0.103 g (0.470 mmol) of (Boc)₂O, 0.010g(0.854 μmol) of DMAP, and 71 μL (0.512 mmol) of TEA. The reactionmixture was stirred at ambient temperature overnight. The reactionmixture was concentrated in vacuo and the residue partitioned betweenCH₂Cl₂ and saturated aqueous NaHCO₃. The layers were separated and theorganics dried over Na₂SO₄, filtered and concentration in vacuo. Thecrude residue was used without further purification.

The preparation described for Compound 4 was repeated except that thecrude residue from above was the reagent employed in place of2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, using the samemolar proportions as before. In this case, the final product obtainedwas a mixture of isomers of tert-butyl2-((8-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrazino[2,3-d]pyridazin-6(5H)-yl)methyl)-5-(trifluoromethyl)-1H-benzo[d]imidazole-1-carboxylate (Compound 15) in 44% yield: ¹H NMR (CDCl₃,300 MHz): δ_(ppm) 9.09-9.06 (m, 4H), 8.26 (s, 1H), 8.03 (m, 1H), 7.83(s, 1H), 7.64-7.50 (m, 3H), 5.96 (s, 4H), 4.04 (s, 4H), 1.76 (s, 18H),1.42 (s, 18H).

Example 12 Preparation of Compound 16

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 15. In thiscase, the final product obtained was2-(8-oxo-7-((5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound 16) in 68% yield: ¹H NMR(DMSO-d6, 300 MHz): δ_(ppm) 9.26 (d, J=2.1 Hz, 1H), 9.21 (d, J=2.1 Hz,1H), 7.88 (s, 1H), 7.69 (d, J=8.4 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 5.67(s, 2H), 4.00 (s, 2H).

Example 13 Preparation of Compound 17.

The preparation described for Compound 4 was repeated except thattert-butyl 2-(chloromethyl)-1H-benzo[d]imidazole-1-carboxylate was thereagent employed in place of2-(chloromethyl)-5-(trifluoromethyObenzo[d]thiazole, using the samemolar proportions as before. In this case, the final product obtainedwas tert-butyl 2-((8-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrazino[2,3-d]pyridazin-6(5H)-yl)methyl)-1H-benzo[d]imidazole-1-carboxylate(Compound 17) in 81% yield: ¹H NMR (CDCl₃, 300 MHz): δ_(ppm) 9.08 (d,J=2.1 Hz, 1H), 9.04 (d, J=2.1 Hz, 1H), 7.92 (d, J=8.7 Hz, 1H), 7.54 (d,J=7.5 Hz, 1H), 7.33-7.22 (m, 2H), 5.95 (s, 2H), 4.04 (s, 2H), 1.74 (s,9H), 1.41 (s, 9H).

Example 14 Preparation of Compound 18.

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 17. In thiscase, the final product obtained was2-(7-((1H-benzo[d]imidazol-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound 18) in 69% yield: : ¹H NMR(DMSO-d6, 300 MHz): δ_(ppm) 9.26 (d, J=2.1 Hz, 1H), 9.21 (d, J=2.1 Hz,1H), 7.56-7.53 (m, 2H), 7.24-7.20 (m, 2H), 5.66 (s, 2H), 4.00 (s, 2H).

Example 15 Preparation of Compound 19.

The preparation described for Compound 4 was repeated except that3-(bromomethyl)-5-chlorobenzo [b]thiophene was the reagent employed inplace of 2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, usingthe same molar proportions as before. In this case, the final productobtained was tert-butyl 2-(7-((5-chlorobenzo[b]thiophen-3-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 19) in 69% yield: ¹H NMR (CDCl₃, 300 MHz): δ_(ppm)9.02 (s, 1H), 8.98 (s, 1H), 8.19 (d, J=1.8 Hz, 1H), 7.73-7.70 (m, 2H),7.30-7.27 (m, 1H), 5.63 (s, 2H), 4.02 (s, 2H), 1.40 (s, 9H).

Example 16 Preparation of Compound 20.

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 19. In thiscase, the final product obtained was2-(7-((5-chlorobenzo[b]thiophen-3-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound 20) in 80% yield: : ¹H NMR(DMSO-d6, 300 MHz): δ_(ppm) 9.19 (d, J=1.8 Hz, 1H), 9.16 (d, J=1.8 Hz,1H), 8.18 (d, J=1.8 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 7.79 (s, 1H), 7.41(dd, J=8.4 Hz, 1.8 Hz , 1H), 5.59 (s, 2H), 3.97 (s, 2H).

Example 17 Preparation of Compound 21.

The preparation described for Compound 4 was repeated except that5-chloro-2-(chloromethyl) benzofuran was the reagent employed in placeof 2-(chloromethyl)-5-(trifluoromethyl) benzo[d]thiazole, using the samemolar proportions as before. In this case, the final product obtainedwas tert-butyl2-(7-((5-chlorobenzofuran-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetate (Compound 21) in 60% yield: ¹H NMR (CDCl₃,300 MHz): δ_(ppm) 9.06 (s, 1H), 9.02 (s, 1H), 7.48 (d, J=2.1 Hz, 1H),7.33 (d, J=8.7 Hz, 1H), 7.19 (dd, J=8.7 Hz, 2.1 Hz, 1H), 6.76 (s, 1H),5.59 (s, 2H), 4.03 (s, 2H), 1.41 (s, 9H).

Example 18 Preparation of Compound 22.

The scheme described in Example 1 to obtain Compound A from Compound 4was carried out, where Compound 4 was replaced with Compound 21. In thiscase, the final product obtained was2-(7-((5-chlorobenzofuran-2-yl)methyl)-8-oxo-7,8-dihydropyrazino[2,3-d]pyridazin-5-yl)acetic acid (Compound 22) in 74% yield: : ¹H NMR(DMSO-d6, 300 MHz): δ_(ppm) 9.22 (d, J=2.1 Hz, 1H), 9.19 (d, J=2.1 Hz,1H), 7.68 (d, J=2.1 Hz, 1H), 7.58 (d, J=8.7 Hz, 1H), 7.30 (dd, J=8.7 Hz,2.1 Hz, 1H), 6.91 (s, 1H), 5.56 (s, 2H), 3.98 (s, 2H).

Example 19 Solubility Study of Compound A Versus Zopolrestat in Buffer

Compound A exhibited superior solubility properties over that ofzopolrestat (shown below) when dissolved in Krebs-Henseleit buffercontaining (in mM) NaCl 118, KCl 4.7, CaCl₂ 2.5, MgCl₂ 1.2, NaHCO₃ 25,glucose 5, palmitate 0.4, bovine serum albumin 0.4, and 70 mU/L insulin.

Compound A, when added to the Krebs-Henseleit buffer, was solublewithout any precipitation or turbidity. In the case of zopolrestat, heatwas applied to render zopolrestat soluble in the Krebs-Henseleit buffer.These results show improved solubility of Compound A over that ofzopolrestat.

Example 20 Solubility Study of Compound A versus zopolrestat in water.

Each sample of Compound A & zopolrestat were placed, separately, inwater (MQW ultra nanopure) at ambient temperature and vortexed for 3minutes. The aliquots were then filtered through a tightly-packed cottonplug (placed in a pipette) to remove any residual solids. The attemptedconcentrations of the respective solutions of Compound A and zopolrestatmade were 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 5.0 mg/mL, and10.0 mg/mL. The pH of the water used was 7.1 (measured electronically byan Orion perpHect LogR Meter Model #370).

The filtered samples were run through a binary gradient (program:5%-100% MeCN over 30 mins) on a LC-MS (Shimadzu LCMS-2010A LiquidChromatography Mass Spectrometer, reverse-phase column). A 5 μL volumeof each sample solution was injected for each run. Each trace containeda peak (for Compound A, TR approx. 14.070 mins; for zopolrestat, TRapprox. 16.666 mins) measured at a wavelength of 254 nm for which therespective parent ion masses were observed.

By comparing the absorbance areas under the peaks corresponding toCompound A & zopolrestat in each water solution made (Table 1), asolubility curve for each compound was generated (FIG. 1). The measureddata, and generated solubility curves, show that Compound A possesses asignificantly greater solubility in pH 7.1 water than Zopolrestat atambient temperature at all concentration.

TABLE 1 Integration of Absorbance Peaks for Various Concentrations ofCompounds. Compound A (mg/mL) Area Zopolrestat (mg/mL) Area 0.05 18111310.05 1039247 0.1 3221003 0.1 1899466 0.5 5814304 0.5 4304556 1 105154301 6129559 5 17216187 5 13314544 10 20397336 10 13362465

Example 21 In Vitro Studies of Compound A Versus Zopolrestat

The reductase activity of Compound A and zopolrestat werespectrophotometrically assayed by following the decrease of NADPH at 25°C. for 4 min as described in Sato, S. (1992), “Rat kidney aldosereductase and aldehyde reductase and polyolproduction in rat kidney” Am.J. Physiol. 263, F799.F805, incorporated by reference herein in itsentirety.

Briefly, the reaction mixture (total volume 1 ml) contained 0.1 mMNADPH, 100 mM substrate (DL-glyceraldehyde or L-xylose) and humanrecombinant aldose reductase (100 mU) in 0.1 M phosphate buffer, pH 6.2.The head to head experiment was carried out in a microplate assay for ARinhibition using D-Glyceraldehyde and NADPH and the absorbance changeswere monitored at 340 nm and % inhibition was calculated for ARIs atconcentrations ranging from 0.1 nM to 100 μM (Table 2 and FIG. 2). Datais presented as mean±standard deviation and is an average of 5 separateruns. The reaction was started by adding the substrate (glyceraldehydeor xylose) as well as the same reaction mixture in which the substratereplaced by deionized water was used as a control. One enzyme unit (U)was defined as the activity consuming 1 μmole of NADPH per min at 25° C.

TABLE 2 Aldose Reductase Activity for Zopolrestat and Compound A. %Inhibition % Inhibition Concentration (zopolrestat) (Compound A)  1 pM11.0 10 pM 29.0 0.1 nM  31.7 ± 5.1 69.0 ± 1.3  1 nM 39.3 ± 4.3 79.6 ±2.4 10 nM 52.2 ± 1.9 80.0 ± 1.8 100 nM  76.7 ± 2.7 89.2 ± 0.7  1 μM 86.3± 3.6 93.6 ± 2.8 10 μM 90.1 ± 3.4 95.3 ± 1.1 100 μM  93.2 ± 3.8 96.2 ±3.3

Results show a significant increase in inhibition potency of Compound Aover that of zopolrestat. The concentration versus inhibition curve forCompound A is shown in FIG. 3, and Compound A shows an IC₅₀ of 28.9picomolar for aldose reductase. Compound A is approximately 100× moreactive than zopolrestat in vitro against aldose reductase.

Example 22 Ex Vivo Studies of Compound A

All rat studies were performed with the approval of the InstitutionalAnimal Care and Use Committee at Columbia University, New York. Thisinvestigation conforms to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health (NIHpublication No. 85-23, 1996; hereby incorporated by reference in itsentirety).

Experiments were performed using an isovolumic isolated rat heartpreparation as as described by Hwang Y C, Sato S, Tsai J Y, Yan S, BakrS, Zhang H, Oates P J, Ramasamy R (2002), “Aldose reductase activationis a key component of myocardial response to ischemia,”Faseb J. 16,243-245 and Ramasamy R, Hwang Y C, Whang J, Bergmann S R (2001),“Protection of ischemic hearts by high glucose is mediated, in part, byGLUT-4,”American Journal of Physiology. 281, H290-297; each of whichhereby incorporated by reference in its entirety.

Male Wistar rats (300.350 g, 3 to 4 months old) were anesthetized with amixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). After deepanesthesia was achieved, hearts were rapidly excised, placed into icedsaline, and retrogradely perfused at 37° C. in a non-recirculating modethrough the aorta at a rate of 12.5 ml/min. Hearts were perfused withmodified Krebs-Henseleit buffer containing (in mM) NaCl 118, KCl 4.7,CaCl₂ 2.5, MgCl₂ 1.2, NaHCO₃ 25, glucose 5, palmitate 0.4, bovine serumalbumin 0.4, and 70 mU/L insulin. The perfusate was equilibrated with amixture of 95% O₂-5% CO₂, which maintained perfusate Po₂>600 mmHg. Leftventricular developed pressure (LVDP) and left ventricular end diastolicpressure (LVEDP) were measured using a latex balloon in the leftventricle. LVDP, heart rate, and coronary perfusion pressure weremonitored continuously on a ADI recorder. All rat hearts subjected to 20min of zero-flow ischemia and 60 min of reperfusion (I/R).

In studies involving the use of aldose reductase inhibitor, hearts wereperfused with modified Krebs-Henseleit buffer containing Compound A(shown below), at a final concentration of 100 nM,10 min prior toischemia and was continued throughout the perfusion protocol. Creatinekinase (CK) release, a marker of myocardial I/R injury, was measured asdescribed by Hwang Y C, Sato S, Tsai J Y, Yan S, Bakr S, Zhang H, OatesP J, Ramasamy R (2002), “Aldose reductase activation is a key componentof myocardial response to ischemia,” Faseb J. 16, 243-245 and RamasamyR, Hwang Y C, Whang J, Bergmann S R (2001), “Protection of ischemichearts by high glucose is mediated, in part, by GLUT-4,”American journalof physiology. 281, H290-297; each of which hereby incorporated byreference in its entirety.

Briefly, isolated perfused hearts were subjected to ischemia reperfusion(I/R) injury and the measures of cardiac injury and cardiac functionmonitored. Creatine kinase (CK) release during reperfusion, a marker ofcardiac ischemic injury, was reduced in rat hearts treated with CompoundA than in untreated hearts (Table 3A). Left ventricular developedpressure (LVDP) recovery was greater in rat hearts treated with CompoundA compared to the untreated hearts after I/R (Table 3B), indicatingimproved functional recovery in Compound A treated hearts.

TABLE 3   A) CK Release (expressed as IU/g wet weight) Untreated Rathearts = 939 ± 146 Compound A treated Rat hearts = 425 ± 63 B) LVDPRecovery (expressed as % of pre-ischemic values) Untreated Rat hearts =48 ± 7 Compound A Treated Rat Hearts = 76 ± 5 ***

Although the invention has been described and illustrated in theforegoing illustrative embodiments, it is understood that the presentdisclosure has been made only by way of example, and that numerouschanges in the details of implementation of the invention can be madewithout departing from the spirit and scope of the invention, which islimited only by the claims that follow. Features of the disclosedembodiments can be combined and rearranged in various ways within thescope and spirit of the invention.

What is claimed is:
 1. A method of treating a cardiovascular disordercomprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound of formula (I)

wherein, R¹ is H; X¹ is N; X² is CH; X³ is CH; X⁴ is N; Y is a C═O, C═S,C═NH, or C═N(C₁-C₄)-alkyl; Z is

A¹ is S; A² is N; and R⁷ through R¹⁰ are independently hydrogen,halogen, cyano, acyl, haloalkyl, haloalkoxy, haloalkylthio,trifluoroacetyl, (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, (C₁-C₄)-alkylthio,(C₁-C₄)-alkylsulfinyl, or (C₁-C₄)-alkylsulfonyl; or two of R⁷ throughR¹⁰ taken together are (C₁-C₄)-alkylenedioxy; or a pharmaceuticallyacceptable salt or solvate thereof
 2. The method of claim 1, wherein Yis C═O; and R⁷ through R¹⁰ are independently hydrogen, halogen, cyano,acyl, haloalkyl, haloalkoxy, haloalkylthio, (C₁-C₄)-alkyl,(C₁-C₄)-alkoxy, (C₁-C₄)-alkylthio, (C₁-C₄)-alkylsulfinyl, or(C₁-C₄)-alkylsulfonyl.
 3. The method of claim 2, wherein R⁷ through R¹⁰are independently hydrogen, halogen, or haloalkyl.
 4. The method ofclaim 1, wherein the compound of formula (I) is

or a pharmaceutically acceptable salt thereof
 5. The method of claim 1,wherein the compound of formula (I) is

or a pharmaceutically acceptable salt or solvate thereof
 6. The methodof claim 1, wherein the compound of formula (I) is

or a pharmaceutically acceptable salt or solvate thereof.
 7. The methodof claim 1, wherein the cardiovascular disorder is atherosclerosis,coronary artery disease, peripheral vascular disease or stroke.
 8. Themethod of claim 4, wherein the cardiovascular disorder isatherosclerosis, coronary artery disease, peripheral vascular disease orstroke.
 9. The method of claim 5, wherein the cardiovascular disorder isatherosclerosis, coronary artery disease, peripheral vascular disease orstroke.
 10. The method of claim 6, wherein the cardiovascular disorderis atherosclerosis, coronary artery disease, peripheral vascular diseaseor stroke.
 11. The method of claim 1, wherein the subject is a human.12. The method of claim 4, wherein the subject is a human.
 13. Themethod of claim 5, wherein the subject is a human.
 14. The method ofclaim 6, wherein the subject is a human.
 15. The method of claim 1wherein the cardiovascular disorder is caused by or related to aldosereductase activity.